Herpes simplex virus (HSV) infection of many cultured cells, such as Vero cells, is initiated by receptor-binding and fusion with the cell surface. A survey of HSV entry into diverse cell lines indicated that Chinese hamster ovary (CHO) and HeLa cells support an endocytic, pH-dependent entry pathway for HSV. In these cell types, but not Vero cells, lysosomotropic agents, such as the weak base ammonium chloride and the ionophore monensin which buffer the low pH of organelles, blocked HSV entry in a concentration-dependent manner. Noncytotoxic concentrations of these agents acted at an early, postbinding step during infection by several strains of both serotypes. CHO cell entry mediated by the HSV cellular receptors HveA, nectin-1 or nectin-2 was blocked by these agents. Enveloped virions were readily detected in vesicles by electron microscopy (EM) at < 5 min post-infection. Depletion of cellular energy, which blocks endocytosis, prevented cellular uptake and infection by HSV in CHO and HeLa cells. Entry into Vero cells was unaffected by energy depletion. Fluorescence microscopy showed that the lysosomotropic agent bafilomycin A1 blocked delivery of incoming virus to the nucleus of HeLa and CHO cells, but not Vero cells. Kinetics of HSV uptake from the cell surface of all three cells were similar regardless of the entry pathway utilized (t1/2, 5-10 min). At 15-20 min p.i., internalized virions recovered from CHO-nectin-1 cells remained infectious, and therefore enveloped. Recovery of infectious virus declined by 30 min, suggesting capsid release into the cytosol (penetration) had occurred. In the presence of bafilomycin, infectious, intracellular virions were recovered for as long as 4 hr p.i., indicating they indeed were trapped in endosomes. We propose that HSV has the capacity to efficiently enter cells by endocytic uptake followed by a low pH-triggered step.